Peptide design guideline

• As many features and their combinations affect immunogen design, machine-learning approaches and protein 3D structure/informatics-based programmes are used to design peptide immunogens.
• UniprotKB provides essential information on protein function, structure and properties.
• Establish domain regions. Domains will be present in other proteins and should be avoided in the sequence of the immunogen as they might increase cross reactivity.
• Avoid complex and inaccessible regions such as alpha helices andbeta sheets. Instead aim for accessible regions of the native protein which are surface-exposed, hydrophilic and flexible.
• Epitopes (antigenic determinants) are preferably located atβ turns or loops with amphipathic helices. The C- and N-termini of proteins are often surface-exposed with a high degree of flexibility.
• Peptide length in general 10–20 amino acids. A long peptide increases immunogenicity, but also increases the chance for cross-reactivity while a short peptide improves the specificity, but may not be immunogenic.
• Avoid long chains of hydrophobic residues. Peptide solubility can be improved by changing the peptide length and/or adding 1–2 hydrophilic amino acid residues to the N- or C-terminus.
• Avoid N-terminal glutamine or aspargine and C-terminal proline or glycine in a peptide.
• Avoid internal cysteine, which can be replaced with serine.
• Avoid multiple (more than three) serine or proline residues in a sequence. Also avoid multiple glutamine residues as this can result in hydrogen bonding between peptides.
• Conservative residues can be used to replace any undesirable residues to improve peptide properties.
• Avoid tripeptide arginine-glycine (RGD motif), small molecule binding sites, biologically active regions and post-translational modification site(s) as they may mask the antibody recognition site.
• A terminal cysteine may be added to the peptide sequence to allow peptide conjugation to carrier proteins. It is added away from the epitope location if known. If the peptide is derived from the N-terminus of the protein the cysteine should be added to the C-terminus of the peptide and vice-versa.

• Peptide Purity

For antibody generation and testing, peptide purity > 70% is enough, however, for biological activity studies, peptide purity > 95% is required. 

• Peptide Amino Acid Composition

Amino acid composition governs every aspect of the peptide’s functionality. Peptide antigens should be designed to contain sequences that are found on the surface of the native protein. Both hydrophobic and hydrophilic residues should be included. In particular, it is favorable for the peptide sequence to incorporate amino acids that are antigenic. On the other hand, Cysteine, methionine, and tryptophan are susceptible to oxidation and side reactions. Peptides containing multiple instances of these residues are also difficult to be obtained in high purity. Sometimes, the peptides are chemically modified or conjugated with specific ligands for desired properties, such as solubility, stability, even purification methods. Additional modifications such as N- and C-termini capping, spacer insertions and amino acid replacement are also performed.

• Peptide Length

Peptide length is a critical factor to consider when synthesizing peptide. The longer the peptide sequence, the more difficult for their synthesis and purification. Longer peptides generally have lower crude peptide purity. Peptide antigens'length to cover 10-15 residues for antibody production.

• Peptide Solubility

Hydrophobicity of each constituted amino acids strongly influences the corresponding peptide’s solubility. By changing the frequency of occurrence of hydrophobic and hydrophilic amino acids, Peptides with high incidences of hydrophobic amino acids includingtryptophan, leucine, valine, methionine, phenylalanine, and isoleucine may not easily dissolve in aqueous solutions. Hydrophobic amino acid content is suggested to be kept below 50%.At least one charged residue should be incorporated within every five amino acids. At physiological pH, arginine, glutamine, aspartic acid, and lysine all have charged side chains. A single conservative replacement or addition of polar residues to the N- or C-terminus may also improve peptide solubility.

• Secondary Structure

During peptide synthesis, beta sheet formation can cause incomplete solvation of the growing peptide, which results in a high incidence of deletion sequences in the final product. Avoid multiple or adjacent residues of valine, isoleucine, tyrosine, phenylalanine, tryptophan, leucine, glutamine, or threonine to prevent beta sheet formation. If the above recommendation cannot be implemented practically, conservative replacement may help by reinserting a glycine or proline at every third residue or replacing glutamine with asparagine, or threonine with serine.